Immunotherapeutic aryl amides

ABSTRACT

Novel aryl amides are inhibitors of tumor necrosis factor alpha and can be used to combat cachexia, endotoxic shock, and retrovirus replication. A typical embodiment is N-benzoyl-3-amino-3-(3&#39;,4&#39;-dimethoxyphenyl)propanamide.

This application is a division of Ser. No. 08/366,618 filed Dec. 30,1994, and U.S. Pat. No. 5,801,195.

BACKGROUND OF THE INVENTION

The present invention relates a method of reducing levels of TNFα in amammal and to compounds and compositions useful therein.

TNFα, or tumor necrosis factor α, is a cytokine which is releasedprimarily by mononuclear phagocytes in response to variousimmunostimulators. When administered to animals or humans it causesinflammation, fever, cardiovascular effects, hemorrhage, coagulation andacute phase responses similar to those seen during acute infections andshock states.

Excessive or unregulated TNFα production has been implicated in a numberof disease conditions. These include endotoxemia and/or toxic shocksyndrome {Tracey et al., Nature 330, 662-664 (1987) and Hinshaw et al.,Circ. Shock 30, 279-292 (1990)}; cachexia {Dezube et al., Lancet,335(8690), 662 (1990)}; and Adult Respiratory Distress Svndrome whereTNFα concentration in excess of 12,000 pg/milliliters have been detectedin pulmonary aspirates from ARDS patients {Nlillar et al., Lancet2(8665), 712-714 (1989)}. Systemic infusion of recombinant TNFα alsoresulted in changes typically seen in ARDS {Ferrai-Baliviera et al.,Archi. Surg.124(12), 1400-1405 (1989)}.

TNFα appears to be involved in bone resorption diseases, includingarthritis where it has been determined that when activated, leukocyteswill produce a bone-resorbing activity, and data suggest that TNFαcontributes to this activity. {Bertolini et al., , Nature 319, 516-518(1986) and Johnson et al., Endocrinology 124(3), 1424-1427 (1989).} Ithas been determined that TNFα stimulates bone resorption and inhibitsbone formation in vitro and in vivo through stimulation of osteoclastformation and activation combined with inhibition of osteoblastfunction. Although TNFα may be involved in many bone resorptiondiseases, including arthritis, the most compelling link with disease isthe association between production of TNFα by tumor or host tissues andmalignancy associated hypercalcemia {Calci. Tissue Int. (US) 46(Suppl.),S3-10 (1990)}. In Graft versus Host Reaction, increased serum TNFαlevels have been associated with major complication following acuteallogenic bone marrow transplants {Holler et al., Blood, 75(4),1011-1016 (1990)}.

Cerebral malaria is a lethal hyperacute neurological syndrome associatedwith high blood levels of TNFα and the most severe complicationoccurring in malaria patients. Levels of serum TNFα correlated directlywith the severity of disease and the prognosis in patients with acutemalaria attacks {Grau et al., N. Engl. J. Med. 320(24), 1586-1591(1989)}.

TNFα also plays a role in the area of chronic pulmonary inflammatorydiseases. The deposition of silica particles leads to silicosis, adisease of progressive respiratory failure caused by a fibroticreaction. Antibody to TNFα completely blocked the silica-induced lungfibrosis in mice {Pignet et al., Nature, 344:245-247 (1990)}. Highlevels of TNFα production (in the serum and in isolated macrophages)have been demonstrated in animal models of silica and asbestos inducedfibrosis {Bissonnette et al., Inflammation 13(3), 329-339 (1989)}.Alveolar macrophages from pulmonary sarcoidosis patients have also beenfound to spontaneously release massive quantities of TNFα as comparedwith macrophages from normal donors {Baughman et al., J. Lab. Clin. Med.115(1), 36-42 (1990)}.

TNFα is also implicated in the inflammatory response which followsreperfusion, called reperfusion injury, and is a major cause of tissuedamage after loss of blood flow {Vedder et al. PNAS. 87, 2643-2646(1990)}. TNFα also alters the properties of endothetial cells and hasvarious pro-coagulant activities, such as producing an increase intissue factor pro-coagulant activity and suppression of theanticoagulant protein C pathway as well as down-regulating theexpression of thrombomodulin {Sherry et al., J. Cell Biol. 107,1269-1277 (1988)}. TNFα has pro-inflammatory activities which togetherwith its early production (during the initial stage of an inflammatoryevent) make it a likely mediator of tissue injury in several importantdisorders including but not limited to, myocardial infarction, strokeand circulatory shock. Of specific importance may be TNFα-inducedexpression of adhesion molecules, such as intercellular adhesionmolecule (ICAM) or endothelial leukocyte adhesion molecule (ELAM) onendothelial cells {Munro et al., Am. J. Path. 135(1), 121-132 (1989)}.

Moreover, it now is known that TNFα is a potent activator of retrovirusreplication including activation of HIV-1. {Duh et al., Proc. Nat. Acad.Sci. 86, 5974-5978 (1989); Poll et al., Proc. Nat. Acad. Sci. 87,782-785 (1990); Monto et al., Blood 79, 2670 (1990); Clouse et al., J.Immunol. 142, 431-438 (1989); Poll et al., AIDS Res. Hum. Retrovirus,191-197 (1992)}. AIDS results from the infection of T lymphocytes withHuman Immunodeficiency Virus (HIV). At least three types or strains ofHIV have been identified, i.e., HIV-1, HIV-2 and HIV-3. As a consequenceof HIV infection, T-cell mediated immunity is impaired and infectedindividuals manifest severe opportunistic infections and/or unusualneoplasms. HIV entry into the T lymphocyte requires T lymphocyteactivation. Other viruses, such as HIV-1, HIV-2 infect T lymphocytesafter T cell activation and such virus protein expression and/orreplication is mediated or maintained by such T cell activation. Once anactivated T lymphocyte is infected with HIV, the T lymphocyte mustcontinue to be maintained in an activated state to permit HIV geneexpression and/or HIV replication. Cytokines, specifically TNFα, areimplicated in activated T-cell mediated HIV protein expression and/orvirus replication by playing a role in maintaining T lymphocyteactivation. Therefore, interference Pith cytokine activity such as byprevention or inhibition of cytokine production, notably TNFα, in anHIV-infected individual aids in limiting the maintenance of T lymphocytecaused by HIV infection.

Monocytes, macrophages, and related cells, such as kupffer and glialcells, have also been implicated in maintenance of the HIV infection.These cells, like T cells, are targets for viral replication and thelevel of viral replication is dependent upon the activation state of thecells. {Rosenberg et al., The Immunopathogenesis of HIV Infection,Advances in Immunology, 57 (1989)}. Cytokines, such as TNFα, have beenshown to activate HIV replication in monocytes and/or macrophages {Poliet al. Proc. Nat. Acad. Sci., 87, 782-784 (1990)}, therefore, preventionor inhibition of cytokine production or activity aids in limiting HIVprogression as stated above for T cells. Additional studies haveidentified TNFα as a common factor in the activation of HIV in vitro andhas provided a clear mechanism of action via a nuclear regulatoryprotein found in the cytoplasm of cells (Osborn, et al., PNAS 86,2336-2340). This evidence suggests that a reduction of TNFα synthesismay have an antiviral effect in HIV infections, by reducing thetranscription and thus virus production.

AIDS viral replication of latent HIV in T cell and macrophage lines canbe induced by TNFα {Folks et al., PNAS 86, 2365-2368 (1989)}. Amolecular mechanism for the virus inducing activity is suggested byTNFα's ability to activate a gene regulatory protein (NFκB) found in thecytoplasm of cells, which promotes HIV replication through binding to aviral regulatory gene sequence (LTR) {Osborn et al., PNAS 86, 2336-2340(1989)}. TNFα in AIDS associated cachexia is suggested by elevated serumTNFα and high levels of spontaneous TNFα production in peripheral bloodmonocytes from patients {Wright et al., J. Immunol. 141(1), 99-104(1988)}.

TNFα has been implicated in various roles with other viral infections,such as the cytomegalia virus (CMV), influenza virus, adenovirus, andthe herpes family of viruses for similar reasons as those noted.

Preventing or inhibiting the production or action of TNFα is, therefore,predicted to be a potent therapeutic strategy for many inflammatory,infectious, immunological or malignant diseases. These include but arenot restricted to septic shock, sepsis, endotoxic shock, hemodynamicshock and sepsis syndrome, post ischemic reperfusion injury, malaria,mycobacterial infection, meningitis, psoriasis, congestive heartfailure, fibrotic disease, cachexia, graft rejection, cancer, autoimmunedisease, opportunistic infections in AIDS, rheumatoid arthritis,rheumatoid spondylitis, osteoarthritis, other arthritic conditions,Crohn's disease, ulcerative colitis, multiple sclerosis, systemic lupuserythrematosis, ENL in leprosy, radiation damage, and hyperoxic alveolarinjury. Efforts directed to the suppression of the effects of TNFα haveranged from the utilization of steroids such as dexamethasone andprednisolone to the use of both polyclonal and monoclonal antibodies{Beutler et al., Science 234, 470-474 (1985); WO 92/11383}.

The nuclear factor κB (NFκB) is a pleiotropic transcriptional activator(Lenardo, et al. Cell 1989, 58, 227-29). NFκB has been implicated as atranscriptional activator in a variety of disease and inflammatorystates and is thought to regulate cytokine levels including but notlinited to TNFα and also to be an activator of HIV transcription(Dbaibo, et. al. J. Biol. Chem. 1993, 17762-66; Duh et al. Proc. Natl.Acad. Sci. 1989, 86, 5974-78; Bachelerie et al., Nature 1991, 350,709-12; Boswas et al., J. Acquired Immune Deficiency Syndrome 1993, 6,778-786; Suzuki et al. Biochem. And Biophys. Res. Comm. 1993, 193,277-83; Suzuki et al., Biochem. And Biophys. Res Comm. 1992, 189,1709-15; Suzuki et al., Biochem. Mol. Bio. Int. 1993, 31(4), 693-700;Shakhov et as 1990, 171, 35-47; and Staal et al. Proc. Natl. Acad. Sci.USA 1990, 87, 9943-47). Thus, inhibition of NFκB binding can regulatetranscription of cytokine gene(s) and through this modulation and othermechanisms be useful in the inhibition of a multitude of disease states.The compounds claimed in this patent can inhibit the action of NFκB inthe nucleus and thus are useful in the treatment of a variety ofdiseases including but not limited to rheumatoid arthritis, rheumatoidspondylitis, osteoarthritis, other arthritic conditions, septic shock,septis, endotoxic shock, graft versus host disease, wasting, Crohn'sdisease, ulcerative colitis, multiple sclerosis, systemic lupuserythrematosis, ENL in leprosy, HIV, AIDS, and opportunistic infectionsin AIDS.

TNFα and NFκB levels are influenced by a reciprocal feedback loop. Asnoted above, the compounds of the present invention affect the levels ofboth TNFα and NFκB. It is not known at this time, however, how thecompounds of the present invention regulate the levels of TNFα, NFκB, orboth.

DETAILED DESCRIPTION

The present invention is based on the discovery that a class ofnon-polypeptide imides more fully described herein appear to inhibit theaction of TNFα.

The present invention pertains to compounds of the formula:

in which Ar is (i) straight, branched, or cyclic, unsubstituted alkyl of1 to 12 carbon atoms; (ii) straight, branched, or cyclic, substitutedalkyl of 1 to 12 carbon atoms; (iii) phenyl; (iv) phenyl substitutedwith one or more substituents each selected independently of the otherfrom the group consisting of nitro, cyano, trifluoromethyl, carbethoxy,carbomethoxy, carbopropoxy, acetyl, carbamoyl, acetoxy, carboxy,hydroxy, amino, substituted amino, alkyl of 1 to 10 carbon atoms, alkoxyof 1 to 10 carbon atoms, or halo; (v) heterocycle; or (vi) heterocyclesubstituted with one or more substituents each selected independently ofthe other from nitro, cyano, trifluoromethyl, carbethoxy, carbomethoxy,carbopropoxy, acetyl, carbamoyl, acetoxy, carboxy, hydroxy, amnino,alkyl of 1 to 10 carbon atoms, alkoxy of 1 to 10 carbon atoms, or halo;

R is −H, alkyl of 1 to 10 carbon atoms, CH₂,OH, CH₂CH₂OH, or CH₂COZwhere Z is alkoxy of 1 to 10 carbon atoms, benzyloxy, or NHR¹ where R¹is H or alkyl of 1 to 10 carbon atoms; and,

Y is i) a phenyl or heterocyclic ring, unsubstituted or substituted oneor more substituents each selected independently one from the other fromnitro, cyano, tri-fluoromethyl, carbethoxy, carbomethoxy, carbopropoxy,acetyl, carbamoyl, acetoxy, carboxy, hydroxy, amino, alkyl of 1 to 10carbon atoms, alkoxy of 1 to 10 carbon atoms, or halo or ii) naphthyl.

A first preferred subclass pertains to compounds in which Ar is phenylsubstituted with two methoxy groups;

R is CH₂CO₂CH₃; and

Y is a phenyl ring, unsubstituted or substituted with one amino group.

Typical compounds of this invention include:

N-benzoyl-3-amino-3-(3′,4′-dimethoxyphenyl)propanamide,

N-benzoyl-3-amino-3-(3′,4′-diethoxyphenyl)propanarnide,

N-benzoyl-3-amino-3-(3′,4′-diethylphenyl)propanarmide,

N-benzoyl-3-amino-3-cyclohexylpropanamide,

N-(3″-aminobenzoyl)-3-amino-3-(3′,4′-die thoxyphenyl)propanarnide,

methyl N-benzoyl-3-amino-3-(3′,4′-diethoxphenyl)propanoate,

methyl N-(3″-methoxybenzoyl)-3-amino-3-(3′,4′-diethoxyphenyl)propanoate,

methyl N-(4″-methoxybenzoyl)-3-amno-3-(3′,4′-diethoxyphenylpropanoate,

N-(3″-m ethoxybenzoyl)-3-amino-3-(3′,4′-diethoxy phenyl)propanamide,

N-(4″-methoxybenzoyl)-3-amino-3-(3′,4′-diethoxyphenyl)propanamide,

methyl N-benzoyl-3-amino-3-(4-trifluorome thylphenyl)propanoate,

methyl N-be nzoyl-3-amino-3-(4-acetylphenyl)propanoate.

The term alkyl as used herein denotes a univalent saturated branched orstraight hydrocarbon chain. Unless otherwise stated, such chains cancontain from 1 to 18 carbon atoms. Representative of such alkyl groupsare methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl,tert-butyl, pentyl, isopentyl, neopentyl, tert-pentyl, hexyl, isohexyl,heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl,pentadecyl, hexadecyl, heptadecyl, octadecyl, and the like. Whenqualified by “lower”, the alkyl group will contain from 1 to 6 carbonatoms. The same carbon content applies to the parent term “alkane” andto derivative terms such as “alkoxy”.

The compounds can be used, under the supervision of qualifiedprofessionals, to inhibit the undesirable effects of TNFα. The compoundscan be administered orally, rectally, or parenterally, alone or incombination with other therapeutic agents including antibiotics,steroids, etc., to a mammal in need of treatment. Oral dosage formsinclude tablets, capsules, dragees, and similar shaped, compressedpharmaceutical forms. Isotonic saline solutions containing 20-100milligrams/milliliter can be used for parenteral administration whichincludes intramuscular, intrathecal, intravenous and intra-arterialroutes of administration. Rectal administration can be effected throughthe use of suppositories formulated from conventional carriers such ascocoa butter.

Dosage regimens must be titrated to the particular indication, the age,weight, and general physical condition of the patient, and the responsedesired but generally doses wil be from about 1 to about 500milligrams/day as needed in single or multiple daily administration. Ingeneral, an initial treatment regimen can be copied from that known tobe effective in interfering with TNFα activity for other ThTA mediateddisease states by the compounds of the present invention. Treatedindividuals will be regularly checked for T cell numbers and T4/T8ratios and/or measures of viremia such as levels of reversetranscriptase or viral proteins, and/or for progression ofcytokine-mediated disease associated problems such as cachexia or muscledegeneration. If no effect is soon following the normal treatmentregimen, then the amount of cytokine activity interfering agentadministered is increased, e.g., by fifty percent a week.

The compounds of the present invention also can be used topically in thetreatment or prophylaxis of topical disease states mediated orexacerbated by excessive TNFα production, respectively, such as viralinfections, such as those caused by the herpes viruses, or viralconjunctivitis, etc.

The compounds also can be used in the veterinary treatment of mammalsother than humans in need of prevention or inhibition of TNFαproduction. TNFα mediated diseases for treatment, therapeutically orprophylactically, in animals include disease states such as those notedabove, but in particular viral infections. Examples include felineimmunodeficiency virus, equine infectious anaemia virus, caprinearthritis virus, visna virus, and maedi virus, as well as otherlentiviruses.

Certain of these compounds possess centers of chirality and can exist asoptical isomers. Both the racemates of these isomers and the individualisomers themselves, as well as diastereoisomers when there are twochiral centers, are within the scope of the present invention. Theracemates can be used as such or can be separated into their individualisomers mechanically as by chromatography using a chiral absorbent.Alternatively, the individual isomers can be prepared in chiral form orseparated chemically from a mixture by forming salts with a chiral acid,such as the individual enantiomers of 10-camphorsulfonic acid, camphoricacid, alpha-bromocamphoric acid, methoxyacetic acid, tartaric acid,diacetyltartaric acid, malic acid, pyrrolidone-5-carboxylic acid, andthe like, and then freeing one or both of the resolved bases, optionallyrepeating the process, so as obtain either or both substantially free ofthe other; i.e., in a form having an optical purity of >95%.

Prevention or inhibition of production of TNFα by these compounds can beconveniently assaved using anti-TNFα antibodies. For example, plates(Nunc Immunoplates, Roskilde, DK) are treated with 5 μg/milliliter ofpurified rabbit anti-TNFα antibodies at 4° C. for 12 to 14 hours. Theplates then are blocked for 2 hours at 25° C. with PBS/0.05% Tweencontaining 5 milligrams/milliliter BSA. After washing, 100 μL ofunknowns as well as controls are applied and the plates incubaed at 4°C. for 12 to 14 hours. The plates are washed and assayed with aconjugate of peroxidase (horseradish) and mouse anti-TNFα monoclonalantibodies, and the color developed with o-phenylenediamine inphosphate-citrate buffer containing 0.012% hydrogen peroxide and read at492 nm.

The compounds can be prepared using methods which are known in generalfor the preparation of imides. General reaction schemes include thereaction of the substituted amine or ammonium with substituted benzoylchloride as illustrated by the formulas:

The following examples will serve to further typify the nature of thisinvention but should not be construed as a limitation in the scopethereof, which scope is defined solely by the appended claims.

EXAMPLE 1

Methyl N-benzoyl-3-amino-3-(3,4-dimethoxyphenyl) propionate. To an icebath cooled stirred suspension of methyl3-amino-3-(3,4-dimethoxyphenyl)propionate hydrochloride (0.689 grams,2.50 mmol) and triethylamine (0.7 milliliters, 5 mmol) in 15 millilitersof tetrahydrofuran was added 0.3 milliliters of benzoyl chloride (2.6mmol). The cooling bath was removed after 15 minutes and the mixturestirred for an additional 45 minutes. The reaction midxture was thendiluted with 15 milliliters of brine and 15 milliliters of water andthen partially concentrated in vacuo to remove the tetrahydrofuran. Thereaction slurry was filtered, the solid air-dried, then dried in vacuo(60° C., <1 mm) to afford 0.86 g (100%) of the product as a whitepowder: ¹H HMR (dmso-d₆,250 MHz) δ 8.84 (d, J=8.3 Hz, 1 H, NH), 7.83 (m,2 H, Ar), 7.60-7.35 (m, 3 H, Ar), 7.06 (s, 1 H, Ar), 6.90 (m, 2 H, Ar),5.50-5.30 (m, 1 H, CHN), 3.75 (s, 3 H, OCH₃), 3.72 (s, 3 H, OCH₃), 3.46(s, 3 H, CO₂CH₃), 3.05-2.75 (m, 2 H, CH₂); ¹³C NMR (dmso-d₆) δ 170.8,165.6, 148.6, 147.9, 134.9, 134.5, 131.2, 128.3, 127.3, 118.5, 111.6,110.6, 55.5, 55.5, 51.4, 49.7, 40.6. Anal. Calcd for C₁₉H₂₁NO₅.Theoretical C, 66.46; H, 6.16; N, 4.08. Found C, 66.22; H, 6.05; N,3.98.

EXAMPLE 2

Methyl N-(3-nitrobenzoyl)-3-amino-3-(3,4-dimethoxyphenyl)propionate. Toan ice bath cooled stirred suspension of methyl3-amino-3-(3,4-dimethoxyphenyl)propionate hydrochloride (1.38 grams,5.00 mmol) and triethylamine (1.5 milliliters, 10.8 mmol) in 10milliliters of tetrahydrofuran was added 3-nitrobenzoyl chloride (0.928grams, 5.00 mmol) in a single portion. A thick slurry resulted. Thecooling bath was removed after 15 minutes, the mixture diluted with 10milliliters of tetrahydrofuran and the mixture stirred for an additionalhour. The reaction mixture was diluted with 50 milliliters of water andthen partially concentrated in vacuo to remove the tetrahydrofuran. Thereaction slurry was filtered, the solid washed with copious amounts ofwater, air-dried, and dried in vacuo (60° C., <1 mm) to afford 1.85grams (95%) of the product as an off white powder: ¹H NMR (CDCl₃,250MHz) δ 8.63 (t, J=1.9 Hz, 1 H), 8.35 (m, 1 H, Ar), 8.20 (m, 1 H, Ar),7.77 (d, J=8 Hz, 1 H, NH), 7.63 (t, J=8.0 Hz, 1 H), 6.95-6.75 (m, 3 H,Ar), 5.86 (m, 1 H, CHCO), 3.85 (s, 3 H, OCH₃), 3.84 (s, 3 H, OCH₃),3.68, (s, 3 H, CO₂CH₃), 3.01 (m, 2 H, CH₂); ¹³C NMR (CDCl₃) δ 172.0,164.1, 149.1, 148.6, 148.2, 135.8, 133.1, 132.7, 129.8, 126.1, 122.0,118.2, 111.2, 109.9, 55.9, 55.8, 52.0, 50.2, 39.5.

EXAMPLE 3

Methyl N-(3-aminobenzoyl)-3-amino-3-(3,4-dimethoxyphenyl)propionate. Toa solution of methylN-(3-nitrobenzoyl)-3-amino-3-(3,4-dimethoxyphenyl)propionate (1.25grams, 3.22 mmol) in a mixture of 150 milliliters of ethyl acetate and75 milliliters of methanol (mixture gently warmed to dissolve all solidand then allowed to cool to room temperature) was added 0.25 grams of10% Pd/C. The mixture was then treated with 60 psi of H₂ for 2.5 hourson a Parr Type Shaker. Reaction progress was monitored by TLC (1/9 ethylacetate/methylene chloride, UV) and was complete after 2.5 hours. Thereaction mixture was filtered through celite to remove catalyst. Thefiltrate was concentrated in vacuo to afford a white solid which wasdried in vacuo (60 ° C., <1 mm) to afford 1.07 grams (93%) of thedesired product: ¹H NMR (dmso-d₆, 250 MHz) δ 8.60 (d, J=8.5 Hz, 1 H,NH), 7.15-6.8 (m, 6 H, Ar), 6.67 (m, 1 H, Ar), 5.40 (m, 1 H, CHCO), 5.24(m 2 H, ArNH₂), 3.75 (s, 3 H, OCH₃), 3.72 (s, 3 H, OCH₃), 3.56 (s, 3 H,CO₂CH₃), 2.95 (dd, J=8.9, 15.4 Hz, 1 H), 2.81 (dd, J=6.3, 15.4 Hz, 1 H);¹³C NMR (dmso-d₆) δ 170.9, 166.4, 148.6, 148.6, 147.8, 135.6, 135.1,128.6, 118.5, 116.4, 114.4, 112.8, 111.6, 110.6, 55.5, 55.5, 51.4, 49.6,40.7.

EXAMPLE 4

Methyl N-(4-nitrobenzoyl)3-amino-3-(3,4-diniethoxyphenyl) propionate. Toan ice bath cooled stirred suspension of methyl3-amino-3-(3,4-dimethoxyphenyl)propionate hydrochloride(1.38 grams, 5.00mmol) and triethylamine (1.5 milliliters, 10.8 mmol) in 25 millilitersof tetrahydrofuran was added 4-nitrobenzoyl chloride (0.928 grams, 5.00mmol) in a single portion. After 13 minutes, the cooling bath wasremoved and the reaction mrixture stirred for 45 minutes. The reactionmixture was then diluted with 50 milliliters of water. The reactionslurry was filtered and the solid washed with water, air-dried, and thendried in vacuo (60° C., <1 mm) to afford 1.86 grams (94%) of the productas a yellow powder: ¹H NMR (CDCl₃/TMS, 250 MHz) δ 8.27 (d, J=8.8 Hz, 2H), 7.98 (d, J=8.8 Hz, 2 H), 7.77 (d, J=8.1 Hz, 1 H, NH), 6.95-6.75 (m,3 H, Ar), 5.55 (m, 1 H, CH), 3.86 & 3.85 (2 s, 6 H, 2 OCH₃), 3.68 (s, 3H, CO₂CH₃), 3.00 (m, 2 H, CH₂); ¹³C NMR (CDCl₃/TMS) δ 172.2, 164.4,149.6, 1491, 148.7, 139.7, 132.6, 128.2, 123.8, 118.1, 111.2, 109.9,55.9, 55.8, 52.0, 50.0, 39.3.

EXAMPLE 5

Methyl N-(4aminobenzoyl)-3-amino-3-(3,4-dimethoxyphenyl)propionate. To asolution of methylN-(3-nitrobenzoyl)-3-amino-3-(3,4-dimethoxyphenyl)propionate (1.25grams, 3.22 mmol) in a mixture of 100 milliliters of ethyl acetate and50 millilters of methanol (mixture gently warmed to dissolve all solidand then allowed to cool to room temperature) was added 0.25 grams of10% Pd/C. The mixture was then treated with 60 psi of H₂ for 2.5 hourson a Parr Type Shaker. Reaction progress was monitored by TLC (1/9 ethylacetate/methylene chloride, UV) and was complete after 2.5 hours. Thereaction mixture was filtered through celite to remove catalyst. Thefiltrate was concentrated in vacuo to afford a white solid which wasdried in vacuo (60 ° C., <1 mm) to afford 1.10 grams (96%) of thedesired product: ¹H NMR (dmso-d₆, 250 MHz) δ 8.32 (d, J=8.5 Hz, 1 H,NH), 7.57 (d, J=8.6 Hz, 1 H, Ar), 7.03 (s, 1 H, Ar), 6.88 (m, 2 H, Ar),6.54 (d, J=8.6 , 2 H, Ar), 5.62 (s, 2 H, NH₂), 5.33 (m, 1 H, CHCO₂),3.74 (s, 3 H, OCH₃), 3.71 (s, 3 H, OCH₃), 3.56 (s, 3 H, CO₂CH₃), 2.94(dd, J=8.8, 15.3 Hz, 1 H), 2.80 (dd, J=6.5, 15.3 , 1 H); ¹³C NMR(dmso-d₆) δ 170.9, 165.5, 151.7, 148.5, 147.8, 135.4, 128.8, 121.1,118.5, 112.5, 111.6, 110.6, 55.5, 55.5, 51.3, 49.4, 40.8.

EXAMPLE 6

Methyl N-(3-methoxybenzoyl)-3-amino-3-(3′,4′-dimethoxyphenyl)propionate. To an ice bath stirred suspension of methyl3-amino-3-(3′,4′-dimethowxyphenyl)propionate hydrochloride (0.689 grams,2.50 mmol) and 0.7 milliliters of triethylamine in 20 milliliters ofanhydrous tetrahydrofuran was added 3-methoxybenzoyl chloride (2.5 mmol)via syringe. After 30 minutes, the reaction mixture was allowed to warmto room temperature and stirred for 1 hour. The reaction mixture wasthen treated with 20 milliliters of water. The tetrahydrofuran wasremoved in vacuo and the resulting mixture extracted with methylenechloride (2 times with 25 milliliters). The combined extracts were driedover sodium sulfate and contracted to afford a thick oil. The crudeproduct was purified by flash chromatography (silica gel, 1.4/8.6 ethylacetate/hexanes) to afford 0.5 grams (56%) as a pale green solid (wax):mp 123.5-125° C; ¹H NMR (CDCl₃/TMS) δ 8.96 (d, J=7.9, 1 H), 8.19 (m, 1H), 7.45 (m, 1 H), 7.12-6.68 (m, 5 H), 5.59 (m, 1 H), 4.00 (s, 3 H,OCH₃), 3.87 (s, 3 H, OCH₃), 3.85 (s, 3 H, OCH₃), 3.63 (s, 3 H, OCH₃),2,96 (m, 2 H, CH₂); ¹³C NMR (CDCl₃/TMS) δ 171.6, 164.4, 157.6, 148.9,148.2,1133.8, 132.8, 132.3, 121.3, 121.2, 118.1, 111.3, 111.2, 109.9.55.8, 55.8, 51.6, 49.7, 40.4; TLC (2/8 ehtyl acetate/hexanes, UV)R_(f)=0.26. Anal. Calcd for C₂₀H₂₃NO₆. Theory C, 64.33; H, 6.21; N,3.75. Found C, 64.31; H, 6.25; N, 3.63.

EXAMPLE 7

Methyl N-nicotinoyl-3-amino-3-(3′, 4′-dimethoxy-phenyl)propionate. To acooled (0° C.) stirred suspension of 3-amino-3-(3′,4′-dimethoxyphenyl)propionate hydrochloride (1.38 grams, 5.0 mmol) andtriethylamine (1.5 milliliters, 10.8 mmol) in 20 milliliters oftetrahydrofuran was added nicotinoyl chloride hydrochloride (0.89 grams,5.0 mmol). The thick slurry was stirred for 15 minutes and then allowedto warm to room temperature and stirring was continued for 2 hours. Thereaction mixture was treated with 20 milliliters of water resulting in abrown colored solution. The tetrahydrofuran was removed in vacuo and theaqueous layer was extracted with methylene chloride (3 times, 25milliliters). The combined extracts were dried over magnesium sulfateand concentrated in vacuo to afford an oil which solidified overnight.The white solid was dried in vacuo (60° C., <1 mm) to afford 0.52 grams(30%) of crude product. The crude product was purified by flashchromatography (silica gel, 5% methanol/methylene chloride) and dried invacuo (60° C., <1 mm) to afford 0.38 grams (22%) of the product as awhite solid: ¹H NMR (CDCl₃) δ 9.10-9.00(m, 1 H), 8.80-8.69(m, 1 H),8.19-8.08(m, 1 H), 7.65-7.31(m , 2 H), 6.96-6.76(m, 3 H), 5.64-5.50(m ,1 H), 3.87(s , 3 H), 3.86(s, 3 H), 3.67(s , 3 H), 3.14-2.37(m , 2 H). ¹³C NMR (CDCl₃) δ 172.1, 164.6, 152.4, 149.2, 148.7, 148.1, 135.0, 132.8,129.9, 123.5, 118.1, 111.3, 111.2, 109.9, 109.8, 55.9, 52.0, 49.8, 39.5.HPLC 99.47%.

EXAMPLE 8

Methyl N-acetyl-3-(3,4-dimethoxyphenyl)propionate. To an ice bath cooledstirred suspension of methyl 3-anino-3-(3,4-dimethoxyphenyl)propionatehydrochloride (1.97 grams, 7.14 mmol) and triethylainine (2.15milliliters, 15.43 mmol) in 30 milliliters of tetrahydrofuran was addedacetyl chloride (0.51 milliliters, 7.14 mmol). The cooling bath wasremoved after 15 minutes and the mixture stirred for an additional 2hours. The reaction mixture was diluted with water (25 milliliters) andwas then partially concentrated in vacuo to remove the tetrahydrofuran.The remaining aqueous mixture was extracted with methylene chloride (3times, 20 milliliters) and the combined organic extracts were dried overmagnesium sulfate. The methylene chloride was removed in vacuo to afford1.40 grams of crude product as an orange oil. The crude product waspurified by flash chromatography (silica gel , 5% methanol/methylenechloride) to afford 1.22 grams of product as an oil which latersolidified, some minor impurities persisted and the solid wasrecrystallized from hexane/ethyl acetate. The white solid was dried invacuo (60° C., <1 mm) to afford 0.81 grams (41%) of product as a whitesolid: ¹H NMR (CDCl₃) δ 6.92-6.79(m, 3 H), 6.56-6.39(m , 1 H),5.45-5.03(m , 1 H), 3.87(s, 3H), 3,86(s, 3 H), 3.63(s , 3 H),3.02-2.75(m , 2 H), 2.02(s , 3 H); ¹³ C NMR (CDCl₃) δ 171.7, 169.2,149.1, 148.5, 133.1, 118.1, 111.2, 110.0, 55.9, 51.8, 49.4, 39.7, 23.4;HPLC 98.63%.

EXAMPLE 9

Tablets, each containing 50 milligrams of active ingredient, can beprepared in the following manner:

Constituents (for 1000 tablets) active ingredient 50.0 grams lactose50.7 grams wheat starch  7.5 grams polyethylene glycol 6000  5.0 gramstalc  5.0 grams magnesium stearate  1.8 grams demineralized water q.s.

The solid ingredients are first forced through a sieve of 0.6 mm meshwidth. The active ingredient, the lactose, the talc, the magnesiumstearate and half of the starch then are mixed. The other half of thestarch is suspended in 40 milliliters of water and this suspension isadded to a boiling solution of the polyethylene glycol in 100milliliters of water. The resulting paste is added to the pulverulentsubstances and the mixture is granulated, if necessary with the additionof water. The granulate is dried overnight at 35° C., forced through asieve of 1.2 mm mesh width and compressed to form tablets ofapproximately 6 mm diameter which are concave on both sides.

EXAMPLE 10

Tablets, each containing 100 milligrams of active ingredient, can beprepared in the following manner:

Constituents (for 1000 tablets) active ingredient 100.0 grams lactose100.0 grams wheat starch  47.0 grams magnesium stearate  3.0 grams

All the solid ingredients are first forced through a sieve of 0.6 mmmesh width. The active ingredient, the lactose, the magnesium stearateand half of the starch then are mixed. The other half of the starch issuspended in 40 milliliters of water and this suspension is added to 100milliliters of boiling water. The resulting paste is added to thepulverulent substances and the mixture is granulated, if necessary withthe addition of water. The granulate is dried overnight at 35° C.,forced through a sieve of 1.2 mm mesh width and compressed to formtablets of approximately 6 mm diameter which are concave on both sides.

EXAMPLE 11

Tablets for chewing, each containing 75 milligrams of active ingredient,can be prepared in the following manner:

Composition (for 1000 tablets) active ingredient  75.0 grams mannitol230.0 grams lactose 150.0 grams talc  21.0 grams glycine  12.5 gramsstearic acid  10.0 grams saccharin  1.5 grams 5% gelatin solution q.s.

All the solid ingredients are first forced through a sieve of 0.25 mmmesh width. The mannitol and the lactose are mixed, granulated with theaddition of gelatin solution, forced through a sieve of 2 mm mesh width,dried at 50° C. and again forced through a sieve of 1.7 mm mesh width.The active ingredient, the glycine and the saccharin are carefullymixed, the mannitol, the lactose granulate, the stearic acid and thetalc are added and the whole is mixed thoroughly and compressed to formtablets of approximately 10 mm diameter which are concave on both sidesand have a breaking groove on the upper side.

EXAMPLE 12

Tablets, each containing 10 milligrams of active ingredient, can beprepared in the following manner:

Composition (for 1000 tablets) active ingredient  10.0 grams lactose328.5 grams corn starch  17.5 grams polyethylene glycol 6000  5.0 gramstalc  25.0 grams magnesium stearate  4.0 grams demineralized water q.s.

The solid ingredients are first forced through a sieve of 0.6 mmn meshwidth. Then the active ingredient, lactose, talc, magnesium stearate andhalf of the starch are intimately mired. The other half of the starch issuspended in 65 milliliters of water and this suspension is added to aboiling solution of the polyethylene glycol in 260 milliliters of water.The resulting paste is added to the pulverulent substances, and thewhole is mixed and granulated, if necessary with the addition of water.The granulate is dried overnight at 35° C., forced through a sieve of1.2 mm mesh width and compressed to form tablets of approximately 10 mmdiameter which are concave on both sides and have a breaking notch onthe upper side.

EXAMPLE 13

Gelatin dry-filled capsules, each containing 100 milligrams of activeingredient, can be prepared in the following manner:

Composition (for 1000 capsules) active ingredient 100.0 gramsmicrocrystalline cellulose  30.0 grams sodium lauryl sulphate  2.0 gramsmagnesium stearate  8.0 grams

The sodium lauryl sulphate is sieved into the active ingredient througha sieve of 0.2 mm mesh width and the two components are intimately mixedfor 10 minutes. The microcrystalline cellulose is then added through asieve of 0.9 mm mesh width and the whole is again intimately mixed for10 minutes. Finally, the magnesium stearate is added through a sieve of0.8 mm width and, after mixing for a further 3 minutes, the mixture isintroduced in portions of 140 milligrams each into size 0 (elongated)gelatin dry-fill capsules.

EXAMPLE 14

A 0.2% injection or infusion solution can be prepared, for example, inthe following manner:

active ingredient 5.0 grams sodium chloride 22.5 grams phosphate bufferpH 7.4 300.0 grams demineralized water to 2500.0 milliliters

The active ingredient is dissolved in 1000 milliliters of water andfiltered through a mricrofilter. The buffer solution is added and thewhole is made up to 2500 milliliters with water. To prepare dosage unitforms, portions of 1.0 or 2.5 milliliters each are introduced into glassampoules (each containing respectively 2.0 or 5.0 milligrams of activeingredient).

We claim:
 1. The method of inhibiting TNFα-activated retrovirusreplication in a mammal which comprises administering thereto andeffective amount of a compound of the formula:

wherein Ar is 3,4-disubstituted phenyl where each substituent isselected independently of the other from the group consisting of nitro,cyano, trifluoromethyl, carbethoxy, carbomethoxy, carbopropoxy, acetyl,carbamoyl, acetoxy, carboxy, hydroxy, amino, alkyl of 1 to 10 carbonatoms, alkoxy of 1 to 10 carbon atoms, and halo; Z is alkoxy of 1 to 10carbon atoms, benzyloxy, amino, or alkylamino of 1 to 10 carbon atoms;and Y is pyridyl, unsubstituted or substituted with one or moresubstituents each selected, independently one from the other, from thegroup consisting of nitro, cyano, trifluoromethyl, carbethoxy,carbomethoxy, carbopropoxy, acetyl, carbamoyl, acetoxy, carboxy,hydroxy, amino, alkyl of 1 to 10 carbon atoms, alkoxy of 1 to 10 carbonatoms, and halo.
 2. The method according to claim 1 wherein Y isunsubstituted pyridyl.
 3. The method according to claim 2 wherein Ar is3,4-dimethoxyphenyl.
 4. The method according to claim 2 wherein Ar is3,4-diethoxyphenyl.
 5. The method of claim 2 wherein Z is alkoxy, amino,or alkylamino.
 6. The method of claim 5 wherein Ar is phenyl substitutedwith two alkoxy groups.
 7. The method of claim 5 wherein Z is OCH₃. 8.The method according to claim 1, wherein the compound is part of apharmaceutical composition effective upon single or multiple dosage toinhibit TNFα.
 9. The method according to claim 7, wherein Ar is phenylsubstituted with two methoxy groups.
 10. The method of reducing levelsof TNFα in a mammal which comprises administering thereto an effectamount of a compound of the formula:

wherein Ar is 3,4-disubstituted phenyl where each substituent isselected independently of the other from the group consisting of nitro,cyano, trifluoromethyl, carbethoxy, carbomethoxy, carbopropoxy, acetyl,carbamoyl, acetoxy, carboxy, hydroxy, amino, alkyl of 1 to 10 carbonatoms, alkoxy of 1 to 10 carbon atoms, and halo; Z is alkoxy of 1 to 10carbon atoms, benzyloxy, amino, or alkylamino of 1 to 10 carbon atoms;and Y is pyridyl, unsubstituted or substituted with one or moresubstituents each selected, independently one from the other, from thegroup consisting of nitro, cyano, trifluoromethyl, carbethoxy,carbomethoxy, carbopropoxy, acetyl, carbamoyl, acetoxy, carboxy,hydroxy, amino, alkyl of 1 to 10 carbon atoms, alkoxy of 1 to 10 carbonatoms, and halo.
 11. The method according to claim 10 wherein Y isunsubstituted pyridyl.
 12. The method according to claim 11 wherein Aris 3,4-dimethoxyphenyl.
 13. The method according to claim 11 wherein Aris 3,4-diethoxyphenyl.
 14. The method of claim 11 wherein Z is alkoxy,amino, or alkylamino.
 15. The method of claim 14 wherein Ar is phenylsubstituted with two alkoxy groups.
 16. The method of claim 14 wherein Zis OCH₃.
 17. The method according to claim 10, wherein the compound ispart of a pharmaceutical composition effective upon single or multipledosage to inhibit TNFα.
 18. The method according to claim 10, wherein Aris phenyl substituted with two methoxy groups.